Crystallographic studies of V44 mutants of Clostridium pasteurianum rubredoxin: effects of side-chain size on reduction potential

Understanding the structural origins of differences in reduction potentials is crucial to understanding how various electron transfer proteins modulate their reduction potentials and how they evolve for diverse functional roles. Here, the high-resolution structures of several Clostridium pasteurianum rubredoxin (Cp Rd) variants with changes in the vicinity of the redox site are reported in order to increase this understanding. Our crystal structures of [V44L] (at 1.8 A resolution), [V44A] (1.6 A), [V44G] (2.0 A) and [V44A, G45P] (1.5 A) Rd (all in their oxidized states) show that there is a gradual decrease in the distance between Fe and the amide nitrogen of residue 44 upon reduction in the size of the side chain of residue 44; the decrease occurs from leucine to valine, alanine or glycine and is accompanied by a gradual increase in their reduction potentials. Mutation of Cp Rd at position 44 also changes the hydrogen-bond distance between the amide nitrogen of residue 44 and the sulfur of cysteine 42 in a size-dependent manner. Our results suggest that residue 44 is an important determinant of Rd reduction potential in a manner dictated by side-chain size. Along with the electric dipole moment of the 43-44 peptide bond and the 44-42 NH--S type hydrogen bond, a modulation mechanism for solvent accessibility through residue 41 might regulate the redox reaction of the Rds.     

Polar or Apolar—The Role of Polarity for Urea-Induced Protein Denaturation

Urea-induced protein denaturation is widely used to study protein folding and stability; however, the molecular mechanism and driving forces of this process are not yet fully understood. In particular, it is unclear whether either hydrophobic or polar interactions between urea molecules and residues at the protein surface drive denaturation. To address this question, here, many molecular dynamics simulations totalling ca. 7 µs of the CI2 protein in aqueous solution served to perform a computational thought experiment, in which we varied the polarity of urea. For apolar driving forces, hypopolar urea should show increased denaturation power; for polar driving forces, hyperpolar urea should be the stronger denaturant. Indeed, protein unfolding was observed in all simulations with decreased urea polarity. Hyperpolar urea, in contrast, turned out to stabilize the native state. Moreover, the differential interaction preferences between urea and the 20 amino acids turned out to be enhanced for hypopolar urea and suppressed (or even inverted) for hyperpolar urea. These results strongly suggest that apolar urea–protein interactions, and not polar interactions, are the dominant driving force for denaturation. Further, the observed interactions provide a detailed picture of the underlying molecular driving forces. Our simulations finally allowed characterization of CI2 unfolding pathways. Unfolding proceeds sequentially with alternating loss of secondary or tertiary structure. After the transition state, unfolding pathways show large structural heterogeneity.     

Modulation of the redox potential of the [Fe(SCys)(4)] site in rubredoxin by the orientation of a peptide dipole.

Rubredoxins (Rds) may be separated into two classes based upon the correlation of their reduction potentials with the identity of residue 44; those with Ala44 have reduction potentials that are approximately 50 mV higher than those with Val44. The smaller side chain volume occupied by Ala44 relative to that occupied by Val44 has been proposed to explain the increase in the reduction potential, based upon changes in the Gly43-Ala44 peptide bond orientation and the distance to the [Fe(SCys)(4)] center in the Pyrococcus furiosus (Pf) Rd crystal structure compared to those of Gly43-Val44 in the Clostridium pasteurianum (Cp) Rd crystal structure. As an experimental test of this hypothesis, single-site Val44 <--> Ala44 exchange mutants, [V44A]Cp and [A44V]Pf Rds, have been cloned and expressed. Reduction potentials of these residue 44 variants and pertinent features of the X-ray crystal structure of [V44A]Cp Rd are reported. Relative to those of wild-type Cp and Pf Rds, the V44A mutation in Cp Rd results in an 86 mV increase in midpoint reduction potential and the [A44V] mutation in Pf Rd results in a 95 mV decrease in midpoint reduction potential, respectively. In the crystal structure of [V44A]Cp Rd, the peptide bond between residues 43 and 44 is approximately 0.3 A closer to the Fe center and the hydrogen bond distance between the residue 44 peptide nitrogen and the Cys42 gamma-sulfur decreases by 0.32 A compared to the analogous distances in the wild-type Cp Rd crystal structure. The results described herein support the prediction that the identity of residue 44 alone determines whether a Rd reduction potential of about -50 or 0 mV is observed.     

Investigations of the thermostability of rubredoxin models using molecular dynamics simulations.

The affects of differences in amino acid sequence on the temperature stability of the three-dimensional structure of the small beta-sheet protein, rubredoxin (Rd), was revealed when a set of homology models was subjected to molecular dynamics simulations at relatively high temperatures. Models of Rd from the hyperthermophile, Pyrococcus furiosus (Pf), an organism that grows optimally at 100 degrees C, were compared to three mesophilic Rds of known X-ray crystal structure. Simulations covering the limits of known Rd thermostabilities were carried out at temperatures of 300 K, 343 K, 373 K, and 413 K. They suggest that Rd stability is correlated with structural dynamics. Because the dynamic behavior of three Pf Rd models was consistently different from the dynamic behavior of the three mesophilic Rd structures, detailed analysis of the temperature-dependent dynamic behavior was carried out. The major differences between the models of the protein from the hyperthermophile and the others were: (1) an obvious temperature-dependent transition in the mesophilic structures not seen with the Pf Rd models, (2) consistent AMBER energy for the Pf Rd due to differences in nonbonded interaction terms, (3) less variation in the average conformations for the Pf Rd models with temperature, and (4) the presence of more extensive secondary structure for the Pf Rd models. These unsolvated dynamics simulations support a simple, general hypothesis to explain the hyperthermostability of Pf Rd. Its structure simplifies the conformational space to give a single minimum accessible over an extreme range of temperatures, whereas the mesophilic proteins sample a more complex conformational space with two or more minima over the same temperature range.     

Structural and thermodynamics characters of isolated &alpha;-syn12 peptide: long-time temperature replica-exchange molecular dynamics in aqueous solution

The structural and thermodynamics characters of α-syn12 (residues 1-12 of the human α-synuclein protein) peptide in aqueous solution were investigated through temperature replica-exchange molecular dynamics (T-REMD) simulations with the GROMOS 43A1 force field. The two independent T-REMD simulations were completed starting from an initial conformational α-helix and an irregular structure, respectively. Each replica was run for 300 ns. The structural and thermodynamics characters were studied based on parameters such as distributions of backbone dihedral angles, free energy surface, stability of folded β-hairpin structure, and favorite conformations. The results showed that the isolated α-syn12 peptide in water adopted four different conformational states: the first state was a β-hairpin ensemble with Turn(9-6) and four hydrogen bonds, the second state was a β-hairpin ensemble with two turns (Turn(9-6) and Turn(5-2)) and three hydrogen bonds, the third state was a disordered structure with both Turn(8-5) and Turn(5-2), and the last state was a π-helix ensemble. Meanwhile, we studied the free energy change of α-syn12 peptide from the unfolded state to the β-hairpin state, which was in good agreement with the experiments and molecular dynamics simulations for some other peptides. We also analyzed the driving force of the peptide transition. The results indicated that the driving forces were high solvent exposure of hydrophobic Leu8 and hydrophobic residues in secondary structure. To our knowledge, this was the first report to study the isolated α-syn12 peptide in water by T-REMD.     

Dynamic Regulation of Phenylalanine Hydroxylase by Simulated Redox Manipulation

Recent clinical studies revealed increased phenylalanine levels and phenylalanine to tyrosine ratios in patients suffering from infection, inflammation and general immune activity. These data implicated down-regulation of activity of phenylalanine hydroxylase by oxidative stress upon in vivo immune activation. Though the structural damage of oxidative stress is expected to be comparably small, a structural rationale for this experimental finding was lacking. Hence, we investigated the impact of side chain oxidation at two vicinal cysteine residues on local conformational flexibility in the protein by comparative molecular dynamics simulations. Analysis of backbone dynamics revealed a highly flexible loop region (Tyr138-loop) in proximity to the active center of phenylalanine hydroxylase. We observed elevated loop dynamics in connection with a loop movement towards the active site in the oxidized state, thereby partially blocking access for the substrate phenylalanine. These findings were confirmed by extensive replica exchange molecular dynamics simulations and serve as a first structural explanation for decreased enzyme turnover in situations of oxidative stress.     

Dependence of Protein Crystal Stability on Residue Charge States and Ion Content of Crystal Solvent

Protein crystallization is frequently induced by the addition of various precipitants, which directly affect protein solubility. In addition to organic cosolvents and long-chain polymers, salts belong to the most widely used precipitants in protein crystallography. However, despite such widespread usage, their mode of action at the atomistic level is still largely unknown. Here, we perform extensive molecular dynamics simulations of the villin headpiece crystal unit cell to examine its stability at different concentrations of sodium sulfate. We show that the inclusion of ions in crystal solvent at high concentration can prevent large rearrangements of the asymmetric units and a loss of symmetry of the unit cell without significantly affecting protein dynamics. Of importance, a similar result can be achieved by neutralizing several specific charged residues suggesting that they may play an active role in crystal destabilization due to unfavorable electrostatic interactions. Our results provide a microscopic picture behind salt-induced stabilization of a protein crystal and further suggest that adequate modeling of realistic crystallization conditions may be necessary for successful molecular dynamics simulations of protein crystals.     

Fast structural dynamics in reduced and oxidized cytochrome c

The sub‐nanosecond structural dynamics of reduced and oxidized cytochrome c were characterized. Dynamic properties of the protein backbone measured by amide ¹⁵N relaxation and side chains measured by the deuterium relaxation of methyl groups change little upon change in the redox state. These results imply that the solvent reorganization energy associated with electron transfer is small, consistent with previous theoretical analyses. The relative rigidity of both redox states also implies that dynamic relief of destructive electron transfer pathway interference is not operational in free cytochrome c.     

Thermal stability of Clostridium pasteurianum rubredoxin: deconvoluting the contributions of the metal site and the protein

To provide a framework for understanding the hyperthermostability of some rubredoxins, a comprehensive analysis of the thermally induced denaturation of rubredoxin (Rd) from the mesophile, Clostridium pasteurianum was undertaken. Rds with three different metals in its M(SCys)4 site (M = Fe3+/2+, Zn2+, or Cd2+) were examined. Kinetics of metal ion release were monitored anaerobically at several fixed temperatures between 40 and 100 degrees C, and during progressive heating of the iron-containing protein. Both methods gave a thermal stability of metal binding in the order Fe2+ < Fe3+ < Zn2+ < Cd2+. The temperature at which half of the iron was released from the protein in temperature ramp experiments was 69 degrees C for Fe2+ Rd and 83 degrees C for Fe3+ Rd. Temperature-dependent changes in the protein structure were monitored by differential scanning calorimetry, tryptophan fluorescence, binding of a fluorescent hydrophobic probe, and 1H NMR. Major but reversible structural changes, consisting of swelling of the hydrophobic core and opening of a loop region, were found to occur at temperatures (50-70 degrees C) much lower than those required for loss of the metal ion. For the three divalent metal ions, the results suggest that the onset of the reversible, lower-temperature structural changes is dependent on the size of the MS4 site, whereas the final, irreversible loss of metal ion is dependent on the inherent M-SCys bond strength. In the case of Fe3+ Rd, stoichiometric Fe3+/cysteine-ligand redox chemistry also occurs during metal ion loss. The results indicate that thermally induced unfolding of the native Cp Rd must surmount a significant kinetic barrier caused by stabilizing interactions both within the protein and within the M(SCys)4 site.     

Protein contributions to redox potentials of homologous rubredoxins: an energy minimization study.

The energetic contributions of the protein to the redox potential in an iron-sulfur protein are studied via energy minimization, comparing homologous rubredoxins from Clostridium pasteurianum, Desulfovibrio gigas, Desulfovibrio vulgaris, and Pyrococcus furiosus. The reduction reaction was divided into 1) the change in the redox site charge without allowing the protein to respond and 2) the relaxation of the protein in response to the new charge state, focusing on the latter. The energy minimizations predict structural relaxation near the redox site that agrees well with that in crystal structures of oxidized and reduced P. furiosus rubredoxin, but underpredicts it far from the redox site. However, the relaxation energies from the energy-minimized structures agree well with those from the crystal structures, because the polar groups near the redox site are the main determinants and the charged groups are all located at the surface and thus are screened dielectrically. Relaxation energies are necessary for good agreement with experimentally observed differences in reduction energies between C. pasteurianum and the other three rubredoxins. Overall, the relaxation energy is large (over 500 mV) from both the energy-minimized and the crystal structures. In addition, the range in the relaxation energy for the different rubredoxins is large (300 mV), because even though the structural perturbations of the polar groups are small, they are very near the redox site. Thus the relaxation energy is an important factor to consider in reduction energetics.     

Effects of environment on the structure of Pyrococcus furiosus rubredoxin: a molecular dynamics study

Molecular dynamics simulations based on a 0.95-A resolution crystal structure of Pyrococcus furiosus have been performed to elucidate the effects of the environment on the structure of rubredoxin, and proteins in general. Three 1-ns simulations are reported here: two crystalline state simulations at 123 and 300 K, and a solution state simulation at 300 K. These simulations show that temperature has a greater impact on the protein structure than the close molecular contacts of the crystal matrix in rubredoxin, although both have an effect on its dynamic properties. These results indicate that differences between NMR solution structures and X-ray crystal structures will be relatively minor if they are done at similar temperatures. In addition, the crystal simulations appears to mimic previous crystallographic experiments on the effects of cryo-temperature on temperature factors, and might provide a useful tool in the structural analysis of protein structures solved at cryo-temperatures.     

Polymorphism of the epidermal growth factor receptor extracellular ligand binding domain: the dimer interface depends on domain stabilization

Epidermal growth factor receptors (EGFRs) and their cytoplasmic tyrosine kinases play important roles in cell proliferation and signaling. The EGFR extracellular domain (sEGFR) forms a dimer upon the binding of ligands, such as epidermal growth factor (EGF) and transforming growth factor α (TGFα). In this study, multiple molecular dynamics (MD) simulations of the 2:2 EGF·sEGFR3-512 dimer and the 2:2 TGFα·sEGFR3-512 dimer were performed in solvent and crystal environments. The simulations of systems comprising up to half a million atoms reveal part of the structural dynamics of which sEGFR dimers are capable. The solvent simulations consistently exhibited a prominent conformational relaxation from the initial crystal structures on the nanosecond time scale, leading to symmetry breaking and more extensive contacts between the two sEGFR monomers. In the crystal control simulation, this symmetry breaking and compaction was largely suppressed by crystal packing contacts. The simulations also provided evidence that the disordered domain IV of sEGFR may act as a stabilizing spacer in the dimer. Thus, the simulations suggest that the sEGFR dimer can take diverse configurations in solvent environments. These biologically relevant conformations of the EGFR signal transduction network can be controlled by contacts among the structural domains of sEGFR and its ligands.     

Quaternary structure of flavorubredoxin as revealed by synchrotron radiation small-angle X-ray scattering

Flavodiiron proteins (FDP) are modular enzymes which function as NO and/or O(2) reductases. Although the majority is composed of two structural domains, the homolog found in Escherichia coli, flavorubredoxin, possesses an extra C-terminal module consisting of a linker and a rubredoxin (Rd) domain necessary for interprotein redox processes. In order to investigate the location of the Rd domain with respect to the flavodiiron structural core, small-angle X-ray scattering was used to construct low-resolution structural models of flavorubredoxin. Scattering patterns from the Rd domain, the FDP core, and full-length flavorubredoxin were collected. The latter two species were found to be tetrameric in solution. Ab initio shape reconstruction and rigid-body modeling indicate a peripheral location for the Rd domains, which appear to have weak contacts with the FDP core. This finding suggests that Rd behaves as an independent domain and is freely available to participate in redox reactions with protein partners.     

Propagation of dynamic changes in barnase upon binding of barstar: an NMR and computational study

NMR spectroscopy and computer simulations were used to examine changes in chemical shifts and in dynamics of the ribonuclease barnase that result upon binding to its natural inhibitor barstar. Although the spatial structures of free and bound barnase are very similar, binding results in changes of the dynamics of both fast side-chains, as revealed by (2)H relaxation measurements, and NMR chemical shifts in an extended beta-sheet that is located far from the binding interface. Both side-chain dynamics and chemical shifts are sensitive to variations in the ensemble populations of the inter-converting molecular states, which can escape direct structural observation. Molecular dynamics simulations of free barnase and barnase in complex with barstar, as well as a normal mode analysis of barnase using a Gaussian network model, reveal relatively rigid domains that are separated by the extended beta-sheet mentioned above. The observed changes in NMR parameters upon ligation can thus be rationalized in terms of changes in inter-domain dynamics and in populations of exchanging states, without measurable structural changes. This provides an alternative model for the propagation of a molecular response to ligand binding across a protein that is based exclusively on changes in dynamics.     

Exploring the molecular basis of heme coordination in human neuroglobin

Neuroglobin (Ngb), a recently discovered ancient heme protein, presents the typical globin fold and is around 20% identical to myoglobin (Mb). In contrast with Mb, however, its heme is hexacoordinated (6c). It is expressed in the nervous system and has been the subject of numerous investigations in the last years, but its function is still unclear. The proposed roles include oxygen transport, reactive oxygen species (ROS) detoxification, hypoxia protection, and redox state sensing. All proposed functions require distal histidine dissociation from the heme to yield a reactive iron. With the aim of understanding the 6c to 5c transition, we have performed molecular dynamics simulations for ferrous Ngb in the 6c, 5c, and oxy states. We also computed free energy profiles associated with the transition employing an advanced sampling technique. Finally, we studied the effect of the redox state of CysCD7 and CysD5, which are known to form a disulfide bridge. Our results show that protein oxidation promotes a stabilization of the pentacoordinated species, thus favoring the protein to adopt the more reactive state and supporting the existence of a molecular mechanism whereby O2 would be released under hypoxic conditions, thereby suggesting an O(2) storage function for Ngb. Taken together, our results provide structural information not available experimentally which may shed light on the protein proposed functions, particularly as a redox sensor.     

Conformational entropy changes upon lactose binding to the carbohydrate recognition domain of galectin-3

The conformational entropy of proteins can make significant contributions to the free energy of ligand binding. NMR spin relaxation enables site-specific investigation of conformational entropy, via order parameters that parameterize local reorientational fluctuations of rank-2 tensors. Here we have probed the conformational entropy of lactose binding to the carbohydrate recognition domain of galectin-3 (Gal3), a protein that plays an important role in cell growth, cell differentiation, cell cycle regulation, and apoptosis, making it a potential target for therapeutic intervention in inflammation and cancer. We used ¹⁵N spin relaxation experiments and molecular dynamics simulations to monitor the backbone amides and secondary amines of the tryptophan and arginine side chains in the ligand-free and lactose-bound states of Gal3. Overall, we observe good agreement between the experimental and computed order parameters of the ligand-free and lactose-bound states. Thus, the ¹⁵N spin relaxation data indicate that the molecular dynamics simulations provide reliable information on the conformational entropy of the binding process. The molecular dynamics simulations reveal a correlation between the simulated order parameters and residue-specific backbone entropy, re-emphasizing that order parameters provide useful estimates of local conformational entropy. The present results show that the protein backbone exhibits an increase in conformational entropy upon binding lactose, without any accompanying structural changes.     

Heterogeneity even at the speed limit of folding: large-scale molecular dynamics study of a fast-folding variant of the villin headpiece

We have performed molecular dynamics simulations on a set of nine unfolded conformations of the fastest-folding protein yet discovered, a variant of the villin headpiece subdomain (HP-35 NleNle). The simulations were generated using a new distributed computing method, yielding hundreds of trajectories each on a time scale comparable to the experimental folding time, despite the large (10,000 atom) size of the simulation system. This strategy eliminates the need to assume a two-state kinetic model or to build a Markov state model. The relaxation to the folded state at 300 K from the unfolded configurations (generated by simulation at 373 K) was monitored by a method intended to reflect the experimental observable (quenching of tryptophan by histidine). We also monitored the relaxation to the native state by directly comparing structural snapshots with the native state. The rate of relaxation to the native state and the number of resolvable kinetic time scales both depend upon starting structure. Moreover, starting structures with folding rates most similar to experiment show some native-like structure in the N-terminal helix (helix 1) and the phenylalanine residues constituting the hydrophobic core, suggesting that these elements may exist in the experimentally relevant unfolded state. Our large-scale simulation data reveal kinetic complexity not resolved in the experimental data. Based on these findings, we propose additional experiments to further probe the kinetics of villin folding.     

Molecular dynamics simulations of Trichomonas vaginalis ferredoxin show a loop-cap transition

The crystal structure of the oxidized Trichomonas vaginalis ferredoxin (Tvfd) showed a unique crevice that exposed the redox center. Here we have examined the dynamics and solvation of the active site of Tvfd using molecular dynamics simulations of both the reduced and oxidized states. The oxidized simulation stays true to the crystal form with a heavy atom root mean-squared deviation of 2 A. However, within the reduced simulation of Tvfd a profound loop-cap transition into the redox center occurred within 6-ns of the start of the simulation and remained open throughout the rest of the 20-ns simulation. This large opening seen in the simulations supports the hypothesis that the exceptionally fast electron transfer rate between Tvfd and the drug metronidazole is due to the increased access of the antibiotic to the redox center of the protein and not due to the reduction potential.